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利用RACE技术和RT-PCR相结合,从欧李[Cerasus humilis(Bge)Sok]果实中克隆获得长度为1798 bp的番茄红素β–环化酶(lycopeneβ-cyclase)基因ChLCYb的cDNA全长序列,开放读码框为1515 bp,编码503个氨基酸。序列分析发现,ChLCYb具有植物LCYb保守区(Plant LCYb conserved region)、FAD/NAD(P)结合区(Dinucleotide-binding signature)、“Cyclase motif 1”、“Cyclase motif 2”和“lycopeneβ-cyclase motif”等典型结构特征,在N–端存在1~84个氨基酸残基组成的转运肽信号序列,在85~106、209~227、373~391和460~480氨基酸区域包含4个跨膜结构域。荧光定量PCR结果表明,ChLCYb在叶中表达量最高,其次是幼果,在根中最低;在欧李果实发育过程中,果皮中ChLCYb表达量高于果肉,果皮中ChLCYb表达量先升高,在花后90 d左右表达量最高,然后呈缓慢下降趋势,而果肉中ChLCYb表达量相对平稳。ChLCYb表达模式与果皮和果肉中β–胡萝卜素含量的积累呈显著正相关(r=0.824和r=0.712,P<0.05)。利用大肠杆菌工程菌体系诱导表达了ChLCYb蛋白,并证实其可催化工程菌株中番茄红素向β–胡萝卜素转化,其转化效率达71.22%。在番茄中超量表达ChLCYb促进果实中番茄红素向β–胡萝卜素的转化和积累,转基因株系L-11号果实中的β–胡萝卜素含量高达692.18μg·g~(-1) DW,是非转基因对照的4.42倍。
The full-length cDNA sequence of lycopene β-cyclase gene ChLCYb was cloned from the fruit of Cerasus humilis (Bge) Sok by RACE and RT-PCR , The open reading frame is 1515 bp, encoding 503 amino acids. Sequence analysis showed that ChLCYb has the plant LCYb conserved region, the FAD / NAD (P) binding region, the “Cyclase motif 1”, the “Cyclase motif 2” and the “ lycopeneβ-cyclase motif ”. The signal sequence of transit peptide consisting of 1 to 84 amino acid residues at the N-terminus contains the peptide sequence of amino acid 85 to 106, 209 to 227, 373 to 391 and 460 to 480 Transmembrane domain. The result of real-time PCR showed that ChLCYb was highest in leaves, followed by young fruit and lowest in roots. During the development of P. plum fruit, the expression of ChLCYb in pericarp was higher than that in pulp, the expression of ChLCYb in pericarp first increased, At 90 days after flowering, the expression level was the highest, then decreased slowly, while the expression level of ChLCYb in the pulp was relatively stable. The expression pattern of ChLCYb was positively correlated with the accumulation of β-carotene in peel and pulp (r = 0.824 and r = 0.712, P <0.05). The expression of ChLCYb protein was induced by Escherichia coli engineering bacteria system, and confirmed that it could catalyze the transformation of lycopene to β-carotene in the engineering strain with the conversion efficiency of 71.22%. Overexpression of ChLCYb in tomato promoted the conversion and accumulation of lycopene to β-carotene in fruit, and the content of β-carotene in the transgenic line L-11 was as high as 692.18μg · g -1 DW 4.42 times of the transgenic control.